Bioluminescent Assay of Microbial ATP in Postmortem Tissues for the Estimation of Postmortem Interval

To study the relationship between changes of microbial ATP in four kinds of murine tis-sues and the postmortem interval （PMI）, healthy SD rats were sacrificed and their muscles, livers, spleens and kidneys were sampled at different postmortem intervals. The concentration of microbial ATP was detected using bioluminescent assay and the data was statistically analyzed. The concentra-tion of microbial ATP in muscle increased with PMI time. The peak appeared at the 7th day after death, and at the 10th day, microbial ATP in muscle tissue increased again. In internal organs, the peaks of microbial ATP were observed at the 8th day after death and the level decreased during 8–10 d. The differences in microbial ATP concentration in liver, spleen and kidney were not statistically significant. During day 0 to day 9 after death, the correlation was best between PMI and microbial ATP in muscle. With PMI as the independent variable, the cubic polynomial regression equation was Y=0.02X3–0.166X2–0.666X＋13.412 （R2=0.989, P〈0.01）. In internal organs, the best correlation was found between PMI and microbial ATP during day 0 to day 10. With PMI as the independent variable, the cubic polynomial regression equation was Y=0.016X3–0.127X2–0.809X＋13.324 （R2=0.986, P〈0.01）. There existed high correlations between PMI and microbial ATP concentration in rat tissues. Since only a small amount of tissue was needed for the detection and the sample was not affected by self-decomposition, the method may extend the time range of PMI estimation.

Reversion of Bioluminescent Bacteria (Mutatox^(TM)) toTheir Luminescent State upon Exposure to OrganicCompounds, Munitions, and Metal Salts

Mutatox is a new genotoxicity bioassay which uses as the endpoint the bioluminescence produced on reversion of a dark strain of the marine bacterium Vibrio fischeri ±S9.Reversion can occur by several mechanisms, including base substitution, frame-shift, SOS induction, and DNA intercalation. For screening, Mutatox provides many advantages over the Salmonella trphimurium (Ames) assay: it requires minimal sterility, employs a shorter incubation period, and does not require culture maintenance. Eighteen organic chemicals (phenol, polynuclear aromatic hydrocarbons, nitrotoluenes, others), Na3PO4, and 4 genotoxic metals (Cu2+, Ni2+, As3+, Cd2+) were tested. Most of the organic compounds positive in S. typhimurium assays were positive in Mutatox. None of the metals was genotoxic in V. fischeri, possibly due to poor uptake from the saline medium

Determination of Binding Parameters of Miramistin to Serum Albumin by the Bioluminescent Method

Bioassay based on the luminescent bacteria was used as a new method for the determination of the kinetic constants of ligand binding to the complexion agent. Miramistin as the cationic surface-active antiseptic and serum blood albumin was taken as a model of interacting agent’s. It was concluded that this approach might have a wide range for applications, namely, in all cases where the ligand is an inhibitor of bacterial luminescence and its complex dose not effect the intensity of this process.

Dynamic Measurement of Intracellular pH Based on Bioluminescent Bacteria

Intracellular pH(pHi)is a fundamental indicator of cellular physiological state,regulating cellular state and function,and has important research values.Although various probes for measuring intracellular pH were available,it is challenging to reflect pHi in real-time and reversible manners.Herein,we developed a whole-cell bioluminescent(BL)probe based on wild type BL bacteria,photobacterium phosphoreum(P.phosphoreum),to determine and image pHi.The dependence of BL intensity of P.phosphoreum on pH values of culture solutions was established.It was found that BL intensity could respond to the change of pH values rapidly and reversibly.We further revealed that P.phosphoreum maintained pH homeostasis in the extracellular pH(pHe)within the range of 5.0–7.0,while intracellular pH homeostasis was destroyed at the alkaline pHe.This method opens up the enormous potential of BL bacteria as an alternative to fluorescence for monitoring and imaging pHi.

Assessment of mercury(II) bioavailability using a bioluminescent bacterial biosensor: Practical and theoretical challenges

Critical methodological challenges in the microbial biosensor approach to assessing Hg（II） bioavailability were evaluated from the perspective of analytical chemists. The main challenge stems from the fact that the chemical speciation of Hg（II） in natural waters exerts a major control on its bioavailability, yet its natural complexation equilibria are extensively altered during conventional bioassays. New data, obtained using a bioluminescent Hg（II）-biosensor, that illustrate these challenges are presented and potential solutions proposed.

Bio-screening and quantification of methyl paraben in vinegar and coconut juice separated by HPTLC

As a widely used food preservative,methyl paraben was experimentally evidenced with serious hormonelike adverse effects.Herein,a high performance thin-layer chromatography platformed bioluminescent bioautography and image analysis for the selective quantification and confirmation of methyl paraben was proposed and validated in vinegar and coconut juice.First,the detectability of the bioautography to the analyte on different layer materials was estimated,revealing that normal silica gel was the best choice.After that,the liquid of sample extract and working solution were separated to overcome the background noises due to co-extracted matrices.The separation result was then coupled to the optimized bioautography,enabling instant and straightforward screening of the targeted conpound.For accurate quantification,bioluninescent inhibition pattern caused by the analyte was processed by image analysis,giving useful sensitivity(LOD>16 mg/kg),precision(RSD<10.1%)and accuracy(spike-recovery rate 76.9%-112.2%).Finally,the suspected result was confirmed by determining its MS fingerprint,further strengthening the reliability of screening.

Bioluminescent nanopaper for rapid screening of toxic substances

Environmental pollution is threatening human health and ecosystems as a result of modern agricultural techniques and industrial progress. A simple nanopaper-based platform coupled with luminescent bacteria Aliivibrio Jischeri （A. Jischeri） as a bio-indicator is presented here, for rapid and sensitive evaluation of contaminant toxicity. When exposed to toxicants, the luminescence inhibition of A. Jischeri-decorated bioluminescent nanopaper （BLN） can be quantified and analyzed to classify the toxicity level of a pollutant. The BLN composite was characterized in terms of morphology and functionality. Given the outstanding biocompatibility of nanocellulose for bacterial proliferation, BLN achieved high sensitivity with a low cost and simplified procedure compared to conventional instruments for laboratory use only. The broad applicability of BLN devices to environmental samples was studied in spiked real matrices （lake and sea water）, and their potential for direct and in situ toxicity screening was demonstrated. The BLN architecture not only survives but also maintains its function during freezing and recycling processes, endowing the BLN system with competitive advantages as a deliverable, ready-to-use device for large-scale manufacturing. The novel luminescent bacteria-immobilized, nanocelullose-based device shows outstanding abilities for toxicity bioassays of hazardous compounds, bringing new possibilities for cheap and efficient environmental monitoring of potential contamination.

Expression and reconstitution of the bioluminescent Ca2+reporter aequorin in human embryonic stem cells,and exploration of the presence of functional IP3 and ryanodine receptors during the early stages of their differentiation into cardiomyocytes

In order to develop a novel method of visualizing possible Ca^(2+) signaling during the early differentiation of h ESCs into cardiomyocytes and avoid some of the inherent problems associated with using fluorescent reporters, we expressed the bioluminescent Ca^(2+) reporter, apo-aequorin, in HES2 cells and then reconstituted active holo-aequorin by incubation with f-coelenterazine. The temporal nature of the Ca^(2+) signals generated by the holo-f-aequorin-expressing HES2 cells during the earliest stages of differentiation into cardiomyocytes was then investigated. Our data show that no endogenous Ca^(2+) transients(generated by release from intracellular stores) were detected in 1–12-day-old cardiospheres but transients were generated in cardiospheres following stimulation with KCl or Ca Cl_2, indicating that holo-f-aequorin was functional in these cells. Furthermore, following the addition of exogenous ATP, an inositol trisphosphate receptor(IP_3R) agonist, small Ca^(2+) transients were generated from day 1 onward. That ATP was inducing Ca^(2+) release from functional IP_3 Rs was demonstrated by treatment with 2-APB, a known IP_3 R antagonist. In contrast, following treatment with caffeine, a ryanodine receptor(Ry R) agonist, a minimal Ca^(2+) response was observed at day 8 of differentiation only. Thus, our data indicate that unlike Ry Rs, IP_3 Rs are present and continually functional at these early stages of cardiomyocyte differentiation.

QM/MM Investigations on the Bioluminescent Decomposition of Coelenterazine Dioxetanone in Obelin

The bioluminescent mechoafism of colenterazine dioxetanone（CZD） in the photoprotein of Obelia（obelin） was investigated by the combined quantum and molecular mechanics（QM/MM） method at TD-DFT level, which involved the real protein environment in decomposition of 1,2-dioxetanones. The anionic decomposition of CZD in （CZD＋H2O）- model can go through a charge transfer（CT） catalyzed asynchronous-concerted process, which can be elucidated by the gradual reversible CT initiated luminescence（GRCTIL） mechanism. The neutral CZD in （CZDH＋H2O） decomposes through an uncatalyzed non-CT biradical process. The anionic decomposition catalyzed by CT, in which the S0/S1 surface ＂double crossing＂ hence has ability to provide high quantum yield of singlet chemiexcitation is thus more possible in bioluminescence of photoprotein.

Hybrid reconstruction framework for model-based multispectral bioluminescence tomography based on Alpha-divergence

Bioluminescence tomography(BLT)is a promising imaging modality that can provide noninvasive three-dimensional visualization information on tumor distribution.In BLT reconstruction,the widely used methods based on regularization or greedy strategy face problems such as over-sparsity,over-smoothing,spatial discontinuity,poor robustness,and poor multi-target resolution.To deal with these problems,combining the advantages of the greedy strategies as well as regularization methods,we propose a hybrid reconstruction framework for model-based multispectral BLT using the support set of a greedy strategy as a feasible region and the Alpha-divergence to combine the weighted solutions obtained by L1-norm and L2-norm regularization methods.In numerical simulations with digital mouse and in vivo experiments,the results show that the proposed framework has better localization accuracy,spatial resolution,and multi-target resolution.

Changes in ATP Levels in Rabbit Blood and Its Application for Estimation of the Postmortem Interval

Summary： Relationship between ATP changes of rabbit blood and postmortem interval （PMI） was studied. Twenty-four healthy rabbits were sacrificed and randomly divided into 3 groups with 8 rab- bits of each group. The bodies of three groups were placed in calorstat at temperature of 15℃, 25℃ and 35℃, respectively. The blood from the right ventricle was sampled through indwelling needle each 4 h until 72 h after death. ATP levels in the blood samples were measured by using ATP fluo- rescence rapid detection technique at different PMIs. Blood ATP levels slightly increased in the early stage after death and then constantly declined at all temperatures （15℃, 25℃, and 35℃）. Cubic polynomial regression equations with log[ATP] as dependent variable （y） and PMI as independent variable （x） at different temperatures and the optimal time period were established as followed： Under 15℃ and during 16-64 h after death, y=-3.027×10^-5x^3＋0.003x^2-0.096x-10.625 （Ra^2=0.992, P〈0.001）; under 25℃ and during 8-56 h after death, y=-2.921×10^-5x^3＋0.002x^2- 0.059x-11.186 （Ra^2=0.989, P〈0.001）; under 35℃ and during 4-36 h after death, y=-9.769×10^-5x^3＋ 0.005x^2 -0.117x-11.166 （Ra^2=0.991, P〈0.001）. The changes in ATP levels in blood collected from right ven- tricle of rabbit cadavers showed relatively stable and regular degradation within 72 h after death at different temperatures.

New record of luminescent Mycena chlorophos(Mycenaceae)from Western Ghats of India

An interesting luminescent Mycena was collected from dead bamboo culms on several occasions from an evergreen forest in Kerala State,India.Detailed morphological and molecular studies with nrITS sequence data confirmed it as Mycena chlorophos.A reappraisal of the species along with comprehensive description,photographs and a discussion with related species is provided.This forms the first record of this species from India.

The 9L^(LUC)/Wistar rat glioma model is not suitable for immunotherapy

The availability of a well-characterized animal brain tumor model will play an important role in identifying treatments for human brain tumors. Wistar rats bearing 9L glioma cells can develop solid, well-circumcised tumors, and may be a useful animal model for the evaluation of various therapeutic approaches for gliosarcomas. In this study, the 9L/Wistar rat glioma model was produced by intracerebral implantation of 9L^LUC glioma cells syngenic to Fischer 344 （F344） rats. Bioluminescence imaging showed that tumors progressively grew from day 7 to day 21 in 9L^LUC/F344 rats, and tumor regression was found in some 9L^LUC/Wistar rats. Hematoxylin-eosin staining verified that intracranial tumors were gliomas. Immunohistochemistry results demonstrated that no CD4- and CD8-positive cells were found in the syngeneic 9L^LUC/F344 model. However, many infiltrating CD4- and CD8-positive cells were observed within the tumors of the 9L^LUC/Wistar model. Our data suggests that compared with 9L/F344 rats, 9L glioma Wistar rats may not be suitable for evaluating brain glioma immunotherapies, even though the model induced an immune response and exhibited tumor regression.

Stem cell homing, tracking and therapeutic efficiency evaluation for stroke treatment using nanoparticles: A systematic review

BACKGROUND Stroke is the second leading cause of death worldwide.There is a real need to develop treatment strategies for reducing neurological deficits in stroke survivors,and stem cell(SC)therapeutics appear to be a promising alternative for stroke therapy that can be used in combination with approved thrombolytic or thrombectomy approaches.However,the efficacy of SC therapy depends on the SC homing ability and engraftment into the injury site over a long period of time.Nonetheless,tracking SCs from their niche to the target tissues is a complex process.AIM To evaluate SC migration homing,tracking and therapeutic efficacy in the treatment of stroke using nanoparticles METHODS A systematic literature search was performed to identify articles published prior to November 2019 that were indexed in PubMed and Scopus.The following inclusion criteria were used:(1)Studies that used in vivo models of stroke or ischemic brain lesions;(2)Studies of SCs labeled with some type of contrast agent for cell migration detection;and(3)Studies that involved in vivo cellular homing and tracking analysis.RESULTS A total of 82 articles were identified by indexing in Scopus and Pub Med.Afterthe inclusion criteria were applied,35 studies were selected,and the articles were assessed for eligibility;ultimately,only 25 studies were included.Most of the selected studies used SCs from human and mouse bone marrow labeled with magnetic nanoparticles alone or combined with fluorophore dyes.These cells were administered in the stroke model(to treat middle cerebral artery occlusion in 74%of studies and for photothrombotic induction in 26%of studies).Fiftythree percent of studies used xenogeneic grafts for cell therapy,and the migration homing and tracking evaluation was performed by magnetic resonance imaging as well as other techniques,such as near-infrared fluorescence imaging(12%)or bioluminescence assays(12%).CONCLUSION Our systematic review provided an up-to-date evaluation of SC migration homing and the efficacy of cellular therapy for stroke treatment in terms of functional and structural improvements in the late stage.

Rapid bioluminescence assay for monitoring rat CES1 activity and its alteration by traditional Chinese medicines

In traditional Chinese medicine herbs(TCM),including Radix Salviae Miltiorrhizae(Danshen),Radix Puerariae Lobatae(Gegen),Radix Angelicae Sinensis(Danggui),and Rhizoma Chuanxiong(Chuanxiong)are widely used for the prevention and treatment of cardiovascular diseases and also often co-administered with Western drugs as a part of integrative medicine practice.Carboxylesterase 1(CES1)plays a pivotal role in the metabolisms of pro-drugs,Since(S)-2-(2-(6-dimethylamino)-benzothiazole)-4,5-dihydrothiazole-4-carboxylate(NLMe)has recently been identified by us as a selective CES1 bioluminescent sensor,we developed a rapid method using this substrate for the direct measurement of CES1 activity in rats.This bioluminescence assay was applied to determine CES1 activity in rat tissues after a two-week oral administration of each of the four herbs noted above.The results demonstrated the presence of CES1 enzyme in rat blood and all tested tissues with much higher enzyme activity in the blood,liver,kidney and heart than that in the small intestine,spleen,lung,pancreas,brain and stomach.In addition,the four herbs showed tissue-specific effects on rat CES1 expression.Based on the CES1 biodistribution and its changes after treatment in rats,the possibility that Danshen,Gegen and Danggui might alter CES1 activities in human blood and kidney should be considered.In summary,a selective and sensitive bioluminescence assay was developed to rapidly evaluate CES1 activity and the effects of orally administered TCMs in rats.

Study on Shigella Detection by ATP Bioluminescence Magnetic Enzyme Immunoassay

[Objective] The research aimed to establish a rapid detection method for Shigella. [Method] Combining immunomagnetic separation technology with ATP bioluminescence technology, a new kind of fast and accurate ATP bioluminescence magnetic enzyme immunoassay technique for Shigella was established. [Result] Using ATP bioluminescence magnetic enzyme immunoassay technique to detect standard solution for Shigella （ATCC 25931 ）, result showed that correlation coefficient between relative light intensity detected by instrument and bacteria concentration detec- ted by culture counting method was 0.981 1. Moreover, relation curve between relative light intensity and Shigella concentration was drawn. [ Conclusion] The method had a high detection speed and accuracy, and could be used for the rapid detection of pathogen in food and environment.

Correlation of in vivo tumor response and singlet oxygen luminescence detection in mTHPC-mediated photodynamic therapy

Excited-state singlet axygen(^(1)O_(2)),generated during photodynamic therapy(PDT),is believed to be the primary cytotoxic agent with a number of clinically approved photosensitizers.Its relative concentration in cells or tissues can be measured directly through its near-infrared(NIR)luminescence emission,which has correlated well with in vitro cell and in triro normal skin treatment responses.Here,its correlation with the response of tumor tiss1e in vito is examined,using the photosensitizer meso-tetralydroxyphenylchlorin(mTHPC)in an animal model comprising luciferase-and green fluorescent protein(GFP)-transduced gliosarcoma grown in a dorsal window chamber.The change in the bioluminescence signal,imaged pre-treatment and at 2,5 and 9 d post treatment,was used as a quantitative measure of the tumor response,which was classified in individual tumors as"non","moderate"and"strong"in order to reduce the variance in the data.Plotting the bioluminescence-based response vs the^(1)O_(2)counts demonstrated clear correlation,indicating tha^(1)O_(2)luminescence provides a valid do-simetric technique for PDT in tumor tissue.

Source reconstruction for bioluminescence tomography via L_(1/2)regularization

Bioluminescence tomography(BLT)is an important noninvasive optical molecular imaging modality in preclinical research.To improve the image quality,reconstruction algorithms have to deal with the inherent ill-posedness of BLT inverse problem.The sparse characteristic of bioluminescent sources in spatial distribution has been widely explored in BLT and many L1-regularized methods have been investigated due to the sparsity-inducing properties of L1 norm.In this paper,we present a reconstruction method based on L_(1/2) regularization to enhance sparsity of BLT solution and solve the nonconvex L_(1/2) norm problem by converting it to a series of weighted L1 homotopy minimization problems with iteratively updated weights.To assess the performance of the proposed reconstruction algorithm,simulations on a heterogeneous mouse model are designed to compare it with three representative sparse reconstruction algorithms,including the weighted interior-point,L1 homotopy,and the Stagewise Orthogonal Matching Pursuit algorithm.Simulation results show that the proposed method yield stable reconstruction results under different noise levels.Quantitative comparison results demonstrate that the proposed algorithm outperforms the competitor algorithms in location accuracy,multiple-source resolving and image quality.

In vivo bioluminescence imaging of hyperglycemia exacerbating stem cells on choroidal neovascularization in mice

AIM： To investigate the influence of hyperglycemia on the severity of choroidal neovascularization（CNV）,especially the involvement of bone marrow-derived cells（BMCs） and underlying mechanisms.·METHODS： BMCs from firefly luciferase（Fluc）/green fluorescent protein（GFP） double transgenic mice were transplanted into C57BL/6J wide-type mice. The recipient mice were injected intraperitoneally with streptozotocin（STZ） daily for 5 consecutive days to induce diabetes mellitus（DM）, followed by CNV laser photocoagulation.The BMCs recruitment in CNV exposed to hyperglycemia was firstly examined in Fluc/GFP chimeric mice by in vivo optical bioluminescence imaging（BLI） and in vitro Fluc assays. The CNV severity was evaluated by H＆E staining and choroidal flatmount. The expression of vascular endothelial growth factor（VEGF） and stromal cell derived factor-1（SDF-1） was detected by Western blot.·RESULTS： BLI showed that the BMCs exerted dynamic effects in CNV model in Fluc/GFP chimeric mice exposed to hyperglycemia. The signal intensity of transplanted Fluc＋GFP＋BMCs in the DM chimeric mice was significantly higher than that in the control chimeric mice with CNV induction at days 5, 7, 14 and 21（121861.67 ±9948.81 vs 144998.33 ±13787.13 photons/second/cm2/sr for control and DM mice, P5d〈0.05; 178791.67±30350.8 vs240166.67 ±22605.3, P7d〈0.05; 124176.67 ±16253.52 vs196376.67 ±18556.79, P14d〈0.05; 97951.60 ±10343.09 vs119510.00 ±14383.76, P21d〈0.05）, which was consistent with in vitro Fluc assay at day 7 [relative light units of Fluc（RLU1）], 215.00±52.05 vs 707.33±88.65, P 〈0.05; RLU1/relative light units of renilla luciferase（RLU2）, 0.90 ±0.17 vs 1.83 ±0.17, P 〈0.05]. The CNVs in the DM mice were wider than those in the control group at days 5, 7, 14 and21（147.83±17.36 vs 220.33±20.17 μm, P5d〈0.05; 212.17 ±24.63 vs 326.83 ±19.49, P7d〈0.05; 163.17 ±18.24 vs265.17 ±20.55, P14d〈0.05; 132.00 ±10.88 vs 205.33 ±12.98,P21d〈0.05）. The average area of CNV in the DM group was larger at 7d（20688.67±3644.96 vs 32218.00±4132.69 μm2,P 〈0.05）. The expression of VEGF and SDF-1 was enhanced in the DM mice.·CONCLUSION： Hyperglycemia promots the vasculo-genesis of CNV, especially the contribution of BMCs,which might be triggered by VEGF and SDF-1 production.

Preparation of recombinant firefly luciferase by a simple and rapid expression and purification method and its application in bacterial detection

A simple and rapid expression and purification method of recombinant firefly luciferase was developed for bacteria detection. A modified luciferase gene from North American firefly Photinus pyralis was cloned into pET28a expression vector and the recombinant protein was produced in Escherichia coli BL21. The recombinant luciferase,equipped with a polyhistidine affinity tag,was purified by immobilized metal ion affinity chromatography (IMAC). The approach generated an abundant expression and an efficient purification of a recombinant luciferase with final yield 1.995mg/L of cell culture. Experiments on the recombinant luciferase also showed that the relative light units (RUL) of the enzyme were 5.8×108,and the specific activity was 2.9×1010 RLU/mg. By applying adenosine triphosphate (ATP) bioluminescence to detection of the coin bacteria using the recombinant protein,the ATP content of bacteria was 9.48×10-16mol/mL,and was identical to the bacteria counts (4500CFU/mL) in order of magnitude. Taken together,our results provided a simple and efficacious method of the preparation of recombinant luciferase,which could be applied in the determination of bacteria via ATP bioluminescence.